Methods for treating plague

ABSTRACT

Provided herein are methods for using compositions that include a fusion protein having a YscF protein domain, a mature F1 protein domain, and a LcrV protein domain. In one embodiment the composition is used to confer immunity to plague, such as pneumonic plague, caused by  Yersinia pestis . In one embodiment, the composition is administered to a mucosal surface, such as by an intranasal route. In one embodiment, the administration to a mucosal surface includes a vector that has a polynucleotide encoding a fusion protein, where the fusion protein includes a YscF protein domain, a mature F1 protein domain, and a LcrV protein domain. The administration is followed by a second administration by a different route, such as an intramuscular route. The second administration includes a fusion protein having the same three domains, and in one embodiment the fusion protein is the same one administered to a mucosal surface.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 62/324,528, filed Apr. 19, 2016, which is incorporated by referenceherein.

GOVERNMENT FUNDING

This invention was made with government support under grant numberAI071634, awarded by the NIH. The government has certain rights in theinvention.

SUMMARY OF THE APPLICATION

Provided herein are methods that include administering a firstcomposition to a subject. The administration is to a mucosal surface,and in one embodiment the administration is by an intranasal route. Thefirst composition includes a vector that has a polynucleotide encoding afusion protein, where the fusion protein includes a YscF protein domain,a mature F1 protein domain, and a LcrV protein domain. The method alsoincludes administering a second composition to the subject by adifferent route, such as an intramuscular route. The second compositionincludes a fusion protein having the same three domains, and in oneembodiment the fusion protein is the same one administered by anintranasal route. In one embodiment, the fusion protein is isolated. Thesecond composition is administrated after the intranasal administration.

In one embodiment, the fusion protein includes at least one linker,where the linker is present between two of the domains. In oneembodiment, the fusion protein includes a His-tag. In one embodiment,the vector is a replication defective adenovirus vector, such as atype-5 (Ad5). In one embodiment, the fusion protein includes the YscFprotein, the mature F1 protein, and the LcrV protein. In one embodiment,the second administration is at least 7 days after the intranasaladministration. In one embodiment, the subject is a human. In oneembodiment, the administering confers immunity to plague, such aspneumonic plague, caused by Yersinia pestis.

As used herein, the term “protein” refers broadly to a polymer of two ormore amino acids joined together by peptide bonds. The term “protein”also includes molecules which contain more than one protein joined by adisulfide bond, or complexes of proteins that are joined together,covalently or noncovalently, as multimers (e.g., dimers, tetramers).Thus, the terms peptide, oligopeptide, and polypeptide are all includedwithin the definition of protein and these terms are usedinterchangeably.

As used herein, the term “polynucleotide” refers to a polymeric form ofnucleotides of any length, either ribonucleotides, deoxynucleotides,peptide nucleic acids, or a combination thereof, and includes bothsingle-stranded molecules and double-stranded duplexes. A polynucleotidecan be obtained directly from a natural source, or can be prepared withthe aid of recombinant, enzymatic, or chemical techniques. In oneembodiment, a polynucleotide is isolated. A polynucleotide can be linearor circular in topology. A polynucleotide can be, for example, a portionof a vector, such as an expression or cloning vector, or a fragment.

As used herein, an “isolated” substance is one that has been removedfrom a cell and many of the proteins, nucleic acids, and other cellularmaterial of its natural environment are no longer present. A substancemay be purified, i.e., at least 60% free, at least 75% free, or at least90% free from other components with which they are naturally associated.Proteins and polynucleotides that are produced by recombinant,enzymatic, or chemical techniques are considered to be isolated andpurified by definition, since they were never present in a cell. Forinstance, a protein, a polynucleotide, or a viral particle can beisolated or purified.

As used herein, the terms “coding region,” “coding sequence,” and “openreading frame” are used interchangeably and refer to a nucleotidesequence that encodes a protein and, when placed under the control ofappropriate regulatory sequences expresses the encoded protein. Theboundaries of a coding region are generally determined by a translationstart codon at its 5′ end and a translation stop codon at its 3′ end.

A “regulatory sequence” is a nucleotide sequence that regulatesexpression of a coding sequence to which it is operably linked.Nonlimiting examples of regulatory sequences include promoters,enhancers, transcription initiation sites, translation start sites,translation stop sites, transcription terminators, and poly(A) signals.The term “operably linked” refers to a juxtaposition of components suchthat they are in a relationship permitting them to function in theirintended manner. A regulatory sequence is “operably linked” to a codingregion when it is joined in such a way that expression of the codingregion is achieved under conditions compatible with the regulatorysequence.

The term “and/or” means one or all of the listed elements or acombination of any two or more of the listed elements.

The words “preferred” and “preferably” refer to embodiments of theinvention that may afford certain benefits, under certain circumstances.However, other embodiments may also be preferred, under the same orother circumstances. Furthermore, the recitation of one or morepreferred embodiments does not imply that other embodiments are notuseful, and is not intended to exclude other embodiments from the scopeof the invention.

The terms “comprises” and variations thereof do not have a limitingmeaning where these terms appear in the description and claims.

It is understood that wherever embodiments are described herein with thelanguage “include,” “includes,” or “including,” and the like, otherwiseanalogous embodiments described in terms of “consisting of” and/or“consisting essentially of” are also provided.

Unless otherwise specified, “a,” “an,” “the,” and “at least one” areused interchangeably and mean one or more than one.

Also herein, the recitations of numerical ranges by endpoints includeall numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2,2.75, 3, 3.80, 4, 5, etc.).

For any method disclosed herein that includes discrete steps, the stepsmay be conducted in any feasible order. And, as appropriate, anycombination of two or more steps may be conducted simultaneously.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows immunoblot analysis of recombinant adenoviruses. Human lungepithelial cells A549 were infected with rAd5 constructs at 1000 v.p.per cell. Host cell lysates were harvested after 24 h p.i. An aliquot ofthe cell lysates was then resolved by SDSPAGE and subjected to Westernblot analysis by using mAb-LcrV antibody. Lane 1: Standard proteinmolecular weight markers in kilo-daltons (kDa). Lanes 2-4: A549 cellsinfected with rAd5-LcrV, rAd5-YFV and Ad5-empty, respectively. Lane 5:Purified rLcrV (50 ng). Lane 6: Purified rYFV (30 ng). The HRP-labeledanti-mouse secondary antibody and ECL Western blotting reagent kit(Millipore, Billerica, Mass.) was used for protein detection.

FIG. 2A-2C shows protection conferred by immunization of mice with thepurified recombinant proteins. Naïve mice (n=40) were immunized witheither the mixture of three recombinant proteins (rYscF, rF1, and rLcrV,25 μg/each) or 45 μg of the corresponding recombinant fusion protein(rYFV) via the i.m route. The antigens were emulsified 1:1 in Alumadjuvant. One primary immunization and two identical boosters were givenon days 0, 15 and 30. Naïve mice received the adjuvant only and servedas a control. Mice were bled 14 days post last immunization and an ELISAwas performed to examine IgG and its isotype antibody titers to the LcrVantigen (FIG. 2A). The P values were in comparison to the indicatedgroups and were based on Two-way ANOVA (IgG1 and IgG2a) with the Tukey'spost hoc correction. The above immunized and control mice were thensplit into two sets and challenged on day 15 post immunization eithersubcutaneously (s.c.) with 8500 LD₅₀ (FIG. 2B) or intranasally (i.n.)with 800 LD₅₀ (FIG. 2C) of the WT CO92. The P values were in comparisonto the control group and were based on Kaplan-Meier Curve Analysis.

FIG. 3A-3C shows immunization routes comparison in mice. Naïve mice(n=40) were either i.m. or i.n. immunized with one dose (8×10⁹ v.p) ofrAd5-LcrV or rAd5-YFV vaccines. Animals received the same dose ofAd5-Empty which was split equally into i.m. injection and i.n.instillation, and served as a control. The above immunized and controlmice were then divided into two sets and challenged on day 15 postimmunization either subcutaneously (s.c.) with 60 LD₅₀ (FIG. 3A) orintranasally (i.n.) with 90 LD₅₀ (FIG. 3B) of the WT CO92. The P valueswere in comparison to the control group and were based on Kaplan-MeierCurve Analysis. Mice were also bled prior to the challenge to evaluateIgG antibody titers and that of its isotypes to LcrV by ELISA (FIG. 3C).The P values were in comparison to the indicated groups and were basedon Two-way ANOVA (IgG1 and IgA) with the Tukey's post hoc correction.

FIG. 4A-4C shows protection conferred by immunization with therecombinant adenoviruses in mice that had pre-existing immunity toadenovirus. To establish pre-existing immunity to adenovirus, naïve mice(n=40) received a single dose (8×10⁹ v.p./100 μl) in both quadriceps (50μl each) of the Ad5-Empty by i.m. injection 30 days prior tovaccination. Naïve mice receiving saline served as a control.Subsequently, mice were i.n. immunized with one dose (8×10⁹ v.p) ofrAd5-LcrV or rAd5-YFV vaccines. Animals received the same dose ofAd5-Empty by i.n. instillation, and served as a negative control. Theabove mice were then divided into two sets and challenged on day 15 postimmunization either subcutaneously (s.c.) with 24 LD₅₀ (FIG. 4A) orintranasally (i.n.) with 21 LD₅₀ (FIG. 4B) of the WT CO92. The P valueswere in comparison to the negative control group and were based onKaplan-Meier Curve Analysis. Mice were also bled prior to the challengeto evaluate IgG antibody titers, titers to its isotypes, and IgA to LcrVby ELISA (FIG. 4C). The P values were in comparison to the indicatedgroups and were based on Two-way ANOVA with the Tukey's post hoccorrection. The asterisks indicated statistical significance compared tothe control (Ad5-Empty) mice for IgA levels by using multiple Student'st-test with the Holm-sidak post hoc test correction.

FIG. 5 shows prime-boost immunization provided better protection to miceagainst lethal WT CO92 aerosol challenge. PreAd-mice (groups of 20) wereeither i.n.-immunized with 8×10⁹ v.p./40 μl of rAd5-YFV alone or in thecombination with 10 μg of rYFV (emulsified 1:1 in Alum adjuvant) i.m.The immunization occurred two weeks apart. Naïve mice immunized witheither 10 μg of rYFV (i.m) or 8×10⁹ v.p./40 μl (i.n.) of rAd5-YFV alonewere used for comparison, and PreAd-mice without further immunizationsserved as a negative control. After 15 days post immunization, mice werechallenged by the aerosol route with WT CO92 at a Dp of 6.34×10⁵ CFU.The P values were in comparison to the negative control group and werebased on Kaplan-Meier Curve Analysis.

FIG. 6A-6C. T cell mediated immune response in mice elicited byimmunization with the rAd5-YFV vaccine alone or in combination withrYFV. PreAd-mice (n=10-25) were either i.n. immunized with 8×10⁹ v.p./40μl of rAd5-YFV alone or in the combination with 10 μg of rYFV(emulsified 1:1 in Alum adjuvant) i.m. The immunizations occurred twoweeks apart. After 15 days post immunization, 20 mice from eachimmunized and 10 from control group were aerosol challenged with WT CO92at a Dp of 4.62×10⁵ CFU. The P values were in comparison to the negativecontrol group or between groups (as indicated by the arrow) and werebased on Kaplan-Meier Curve Analysis (FIG. 6A). On day 15 post lastimmunization, T cells were isolated separately from the spleens ofremaining unchallenged 5 mice in each immunized group. The isolated Tcells were co-cultured with γ-irradiated APCs pulsed or un-pulsed withF1-V fusion protein (100 μg/ml). The IFN-γ producing T cells weremeasured after 2 days of incubation with the APCs by using theenzyme-linked immunospot (Elispot) assay (FIG. 6B). T cell proliferationwas assessed by measuring incorporation of [³H] thymidine on day 3 ofco-culture with the APCs (FIG. 6C). The arithmetic means±standarddeviations were plotted. Data were analyzed by using Two-way ANOVA withthe Tukey's post hoc correction. The statistical significance wasindicated by asterisks in comparison of the pulsed and un-pulsed T cellswithin each group or displayed by a horizontal line with the P value.

FIG. 7A-7C shows antibody responses in mice elicited by immunizationwith the rAd5-YFV vaccine alone or in combination with rYFV. Mice fromdifferent groups (FIG. 6A-6C) were also bled 15 days post immunization,and an ELISA was performed to examine IgG antibody titers, its isotypes,and IgA to the F1 (FIG. 7A), LcrV (FIG. 7B) and YscF (FIG. 7C),respectively. The P values were in comparison to the indicated groupsand based on Two-way ANOVA with the Tukey's post hoc correction. Theasterisks indicated statistical significance compared to the control(Ad5-Empty) mice for IgA levels by using multiple Student's t-test withthe Holm-sidak post hoc test correction.

FIG. 8A-8B-02 shows immunization of mice with the rAd5-YFV vaccine aloneor in combination with rYFV provided protection against lethal primaryaerosol and subsequent intranasal WT CO92 challenges. PreAd-mice (n=10)were either i.n.-immunized with 8×10⁹ v.p./40 μl of rAd5-YFV alone or inthe combination with 10 μg of rYFV (emulsified 1:1 in Alum adjuvant)i.m. The immunizations occurred two weeks apart. PreAd-mice injectedwith Ad5-Empty served as a negative control. After 15 days postimmunization, mice were first challenged with aerosolized WT CO92 at aDp of 4.62×10⁵ CFU. After 32 days of the initial aerosol challenge, thesurvivals from the immunized groups along with five age-matcheduninfected naïve mice were infected with 100 LD₅₀ of WT CO92 luc2 strainby the i.n. route. The deaths were recorded for the initial aerosol andthen the subsequent intranasal challenge, and the percentages ofsurvival were plotted (FIG. 8A). The P values were in comparison to thecontrol group for each challenge and were based on Kaplan-Meier CurveAnalysis. The animals were also imaged by IVIS for bioluminescence onday 3 after WT CO92 luc2 strain i.n. challenge (FIGS. 8B-01 and 8B-02).Panel B-I represented infected naïve mice as i.n. challenge control andthe very right animal in this panel was uninfected image control. PanelB-II, animals immunized with the prime-boost strategy, and panel B-III,animals immunized with rAd5-YFV vaccine alone. The bioluminescence scaleis within the figures and ranged from most intense (top of range) toleast intense (bottom of range).

FIG. 9 shows the rAd5-YFV vaccine in combination with rYFV providedprotection to NHPs with pre-existing adenovirus immunity against lethalaerosol challenge of WT CO92. To induce pre-existing adenovirusimmunity, four NHPs were injected in the quadriceps muscle with 5×10¹⁰v.p. of Ad5-Empty (day 0). On day 30, these NHPs were immunized by theintranasal route with 1×10¹¹ v.p. of rAd5-YFV, followed by 50 μg of rYFVboost (emulsified 1:1 in Alum adjuvant) via the i.m. route on day 42.Another four NHPs received saline only (without immunization) and servedas a control. On day 85, the NHPs were challenged with WT CO92 by theaerosol route with a Dp ranging from 1.32 to 8.08×10⁷ CFU. The animalswere euthanized when reached a clinical score ≧8 or at the terminationof the experiment, and percentage of survival was plotted. The P valueswere in comparison to the NHP control group and are based onKaplan-Meier Curve Analysis.

FIG. 10A-10C shows CT scans. NHPs were subjected to CT scan on day 42(naïve and vaccinated) (FIG. 10A) and on day 88 (3 days post WT CO92challenge) for the control NHPs (FIG. 10B) or day 167 (82 days post WTCO92 challenge) (FIG. 10C) for the immunized ones. The coronal andsagittal images of the lungs and their surrounding areas fromrepresenting NHPs were shown with the resolution of 512×512 pixels. Theimage sharpness was optimized to soft tissue. The arrows indicatedconsolidation patches in the lungs of a representative infected controlNHP.

FIG. 11A-11B shows hematologic analysis. Blood samples of immunized(FIG. 11A) and unimmunized control (FIG. 11B) NHPs were collected fromthe femoral veins and analyzed on the day of challenge with WT CO92 andon days 3 and 6 post challenge (days 88 and 91 post immunization andchallenge) by using a Drew Scientific Hemavet 950 hematology system.WBC: white blood cells; NE: neutrophils; LY: lymphocytes. The arithmeticmeans±standard deviations of the cell counts/μl were plotted. The dottedlines indicated the physiological ranges for each of the correspondingparameters measured.

FIG. 12A-12E shows antibody responses in NHPs immunized with therAd5-YFV vaccine in combination of rYFV. Four randomly selected NHPswere injected in the quadriceps muscle with 5×10¹⁰ v.p. of Ad5-Empty toinduce pre-existing immunity (day 0). On day 30, these NHPs wereimmunized by the intranasal route with 1×10¹¹ v.p. of rAd5-YFV, followedby 50 μg of rYFV boost (emulsified 1:1 in Alum adjuvant) via the i.m.route on day 42. Another four NHPs received saline only (withoutimmunization) and served as a control. On day 85, the NHPs werechallenged with WT CO92 by the aerosol route. Blood samples werecollected from the femoral veins of NHPs at various time points duringthe experiment. The total IgG titers to Ad5 (FIG. 12A), F1 (FIG. 12B),LcrV (FIG. 12C), and YscF (FIG. 12D) as well as IgA titers to LcrV (FIG.12E) on days 42, 56, and 85 were evaluated by ELISA. The P values werein comparison to the indicated groups and were based on Two-way ANOVAwith the Tukey's post hoc correction. The asterisks indicatedstatistical significance compared to the control (Ad5-Empty) mice byusing multiple Student's t-test with the Holm-sidak post hoc testcorrection.

FIG. 13A-13E shows antibody responses of vaccinated NHPs after WT CO92aerosol challenge. Four randomly selected NHPs were injected in thequadriceps muscle with 5×10¹⁰ v.p. of Ad5-Empty to induce pre-existingimmunity (day 0). On day 30, these NHPs were immunized by the intranasalroute with 1×10¹¹ v.p. of rAd5-YFV, followed by 50 μg of rYFV boost(emulsified 1:1 in Alum adjuvant) via the i.m. route on day 42. Anotherfour NHPs received saline only (without immunization) and served as acontrol. On day 85, the NHPs were challenged with WT CO92 by the aerosolroute. Blood samples were collected from the femoral veins of NHPs atvarious time points during the experiment from the immunized NHPs. Thetotal IgG titers to Ad5 (FIG. 13A), F1 (FIG. 13B), LcrV (FIG. 13C), andYscF (FIG. 13D) as well as total IgA titers to LcrV (FIG. 13E) on days85, 98 and 112 were evaluated by ELISA. Days 98 and 112 represented 14and 28 days post WT CO92 challenge after immunization.

FIG. 14 shows histopathological analysis of tissues collected from NHPafter WT CO92 aerosol challenge. Lungs, pleura, mediastinal lymph nodes,liver and the spleen tissues were collected from the control (3 or 4 daypost WT CO92 challenge) and immunized NHPs (82 days post WT CO92challenge) after euthanization and processed for histopathologicalanalysis. The inset from lungs revealed the presence of coccobacilli,presumptively Y. pestis, by Gram staining. The magnification of eachimage is indicated.

FIG. 15-01-15-04 shows protein sequences and examples of nucleotidesequences encoding the proteins.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Provided herein are methods for using a fusion protein. The fusionprotein includes at least three protein domains. The three domains are aYscF protein domain, a mature F1 protein domain, and a LcrV proteindomain. A fusion protein can be isolated, and optionally purified.

An example of a YscF protein domain is depicted at SEQ ID NO:2. Otherexamples of YscF protein domains include those having sequencesimilarity with the amino acid sequence of SEQ ID NO:2.

An example of a mature F1 protein domain is depicted at SEQ ID NO:4.Other examples of mature F1 protein domains include those havingsequence similarity with the amino acid sequence of SEQ ID NO:4.

An example of a LcrV protein domain is depicted at SEQ ID NO:6. Otherexamples of LcrV protein domains include those having sequencesimilarity with the amino acid sequence of SEQ ID NO:6.

An example of a fusion protein is depicted at SEQ ID NO:8. The fusionprotein depicted at SEQ ID NO:8 includes, from amino-terminal tocarboxy-terminal end, a YscF domain, a mature F1 domain, followed by aLcrV domain; however, a fusion protein can include the three domains inany order. Thus, other fusion proteins have the domains in the order of,from amino-terminal to carboxy-terminal end, a LcrV domain, a YscFdomain, followed by a mature F1 domain; a LcrV domain, a mature F1domain, followed by a YscF domain; a YscF domain, a LcrV domain,followed by a mature F1 domain; a mature F1 domain, a YscF domain,followed by a LcrV domain; and a mature F1 domain, a LcrV domain,followed by a YscF domain. Other examples of a fusion protein includethose having sequence similarity with the amino acid sequence of SEQ IDNO:8, and those having sequence similarity with any other fusion proteindescribed herein.

A fusion protein described herein has immunological activity.“Immunological activity” refers to the ability of a protein to elicit animmunological response in a subject. An immunological response to aprotein is the development in a subject of a cellular and/orantibody-mediated immune response to the protein. Usually, animmunological response includes but is not limited to one or more of thefollowing effects: the production of antibodies, B cells, helper Tcells, suppressor T cells, and/or cytotoxic T cells, directed to anepitope or epitopes of the protein. “Epitope” refers to the site on anantigen to which specific B cells and/or T cells respond so thatantibody is produced. The immunological activity may be protective.“Protective immunological activity” refers to the ability of a proteinto elicit an immunological response in a subject that prevents orinhibits infection by a Yersinia spp., such as Yersinia pestis. Whethera protein has protective immunological activity can be determined bymethods known in the art such as, for example, the methods described inExample 1. For example, a protein described herein, or combination ofproteins described herein, protects a subject against challenge with aYersinia pestis.

Sequence similarity of two proteins can be determined by aligning theresidues of the two proteins (for example, a candidate protein domainand a reference protein, e.g., a YscF protein domain such as SEQ IDNO:2, a mature F1 protein domain such as SEQ ID NO:4, a LcrV proteindomain such as SEQ ID NO:6, or a fusion protein such as SEQ ID NO:8) tooptimize the number of identical amino acids along the lengths of theirsequences; gaps in either or both sequences are permitted in making thealignment in order to optimize the number of identical amino acids,although the amino acids in each sequence must nonetheless remain intheir proper order. A reference protein may be a protein describedherein. A candidate protein is the protein being compared to thereference protein. A candidate protein may be isolated, for example,from a microbe such as a Yersinia pestis, or can be produced usingrecombinant techniques, or chemically or enzymatically synthesized. Whenthe candidate protein domain is present as part of a fusion protein,only those amino acids of the protein domain are compared with areference protein. For instance, if the candidate protein is YscF and ispart of a fusion protein, only those residues of the YscF domain of thefusion protein are aligned with a reference protein.

Unless modified as otherwise described herein, a pair-wise comparisonanalysis of amino acid sequences can be carried out using the Blastpprogram of the BLAST 2 search algorithm, as described by Tatiana et al.,(FEMS Microbiol Lett, 174, 247-250 (1999)), and available on theNational Center for Biotechnology Information (NCBI) website. Thedefault values for all BLAST 2 search parameters may be used, includingmatrix=BLOSUM62; open gap penalty=11, extension gap penalty=1, gapx_dropoff=50, expect=10, wordsize=3, and filter on. Alternatively,proteins may be compared using the BESTFIT algorithm in the GCG package(version 10.2, Madison Wis.).

In the comparison of two amino acid sequences, structural similarity maybe referred to by percent “identity” or may be referred to by percent“similarity.” “Identity” refers to the presence of identical aminoacids. “Similarity” refers to the presence of not only identical aminoacids but also the presence of conservative substitutions. Aconservative substitution for an amino acid in a protein describedherein may be selected from other members of the class to which theamino acid belongs. For example, it is known in the art of proteinbiochemistry that an amino acid belonging to a grouping of amino acidshaving a particular size or characteristic (such as charge,hydrophobicity and hydrophilicity) can be substituted for another aminoacid without altering the activity of a protein, particularly in regionsof the protein that are not directly associated with biologicalactivity. For example, nonpolar (hydrophobic) amino acids includealanine, leucine, isoleucine, valine, proline, phenylalanine,tryptophan, and tyrosine. Polar neutral amino acids include glycine,serine, threonine, cysteine, tyrosine, asparagine and glutamine. Thepositively charged (basic) amino acids include arginine, lysine andhistidine. The negatively charged (acidic) amino acids include asparticacid and glutamic acid. Conservative substitutions include, for example,Lys for Arg and vice versa to maintain a positive charge; Glu for Aspand vice versa to maintain a negative charge; Ser for Thr so that a free—OH is maintained; and Gln for Asn to maintain a free —NH2.

Guidance concerning how to make phenotypically silent amino acidsubstitutions is provided in Bowie et al. (1990, Science,247:1306-1310), wherein the authors indicate proteins are surprisinglytolerant of amino acid substitutions. For example, Bowie et al. disclosethat there are two main approaches for studying the tolerance of aprotein sequence to change. The first method relies on the process ofevolution, in which mutations are either accepted or rejected by naturalselection. The second approach uses genetic engineering to introduceamino acid changes at specific positions of a cloned gene and selects orscreens to identify sequences that maintain functionality. As stated bythe authors, these studies have revealed that proteins are surprisinglytolerant of amino acid substitutions. The authors further indicate whichchanges are likely to be permissive at a certain position of theprotein. For example, most buried amino acid residues require non-polarside chains, whereas few features of surface side chains are generallyconserved. Other such phenotypically silent substitutions are describedin Bowie et al, and the references cited therein.

Guidance on how to modify the amino acid sequences of the proteindomains disclosed herein can also be obtained by producing a proteinalignment of a reference protein (e.g., SEQ ID NO:2, SEQ ID NO:4, or SEQID NO:6) with other related polypeptides. For instance, the referenceprotein SEQ ID NO:2 can be aligned in a multiple protein alignment withother YscF proteins. Such an alignment shows the locations of residuesthat are identical between each of the proteins, the locations ofresidues that are conserved between each of the proteins, and thelocations of residues that are not conserved between each of theproteins. By reference to such an alignment, the skilled person canpredict which alterations to an amino acid sequence are likely to modifyactivity, as well as which alterations are unlikely to modify activity.Methods for producing multiple protein alignments are routine, andalgorithms such as ClustalW (Larkin et al., 2007, ClustalW and ClustalXversion 2, Bioinformatics 23(21): 2947-2948) and Clustl Omega (Sieverset al., 2011, Molecular Systems Biology 7: 539, doi:10.1038/msb.2011.75;Goujon et al., 2010, Nucleic acids research 38 (Suppl 2):W695-9,doi:10.1093/nar/gkq313).

Thus, as used herein, a candidate protein domain useful in the methodsdescribed herein includes those with at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% aminoacid sequence similarity, or complete identity to a reference amino acidsequence.

Alternatively, as used herein, a candidate protein useful in the methodsdescribed herein includes those with at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% aminoacid sequence similarity, or complete identity to the reference aminoacid sequence.

In one embodiment, a fusion protein described herein includes a linkerbetween one or more the protein domains. A linker is an amino acidsequence that joins protein domains in a fusion protein. A linker can beflexible or rigid, and in one embodiment is flexible. In one embodiment,a linker can be at least 3, at least 4, at least 5, or at least 6 aminoacids in length. It is expected that there is no upper limit on thelength of a linker used in a fusion protein described herein; however,in one embodiment, a linker is no greater than 10, no greater than 9, nogreater than 8, or no greater than 7 amino acids in length. Many linkersare known to a skilled person (see Chen et al. 2013, Adv, Drug Deliv.Rev., 65(10):1357-1369). Specific examples of linkers include GGGS (SEQID NO:11). In one embodiment, a fusion protein can include more than onetype of linker, e.g., one type of linker between a YscF protein domainand a mature F1 protein domain, and another type of linker between amature F1 protein and a LcrV protein. In one embodiment, a fusionprotein can include more than one linker between two protein domains,e.g., two GGGS (SEQ ID NO:11) linkers or three GGGS (SEQ ID NO:11)linkers between a YscF protein domain and a mature F1 protein domain. Anexample of a fusion protein having three GGGS (SEQ ID NO:11) linkersbetween the domains is depicted at SEQ ID NO:10 (the amino acidscorresponding to the linkers are underlined). This fusion proteinincludes, from amino-terminal to carboxy-terminal end, a YscF domain, amature F1 domain, followed by a LcrV domain, with three GGGS linkersbetween the YscF domain and the mature F1 domain, and three GGGS linkersbetween the mature F1 domain and the LcrV domain.

A fusion protein as described herein also can be designed to include oneor more additional sequences such as, for example, the addition ofC-terminal and/or N-terminal amino acids. In one embodiment, additionalamino acids may facilitate purification by trapping on columns or use ofantibodies. Such additional amino acids include, for example,histidine-rich tags that allow purification of proteins on nickelcolumns.

Also provided are polynucleotides encoding a fusion protein describedherein that includes at least three protein domains. Given the aminoacid sequence of a fusion protein described herein that includes atleast three protein domains, a person of ordinary skill in the art candetermine the full scope of polynucleotides that encode that amino acidsequence using conventional, routine methods. The class of nucleotidesequences encoding a selected protein sequence is large but finite, andthe nucleotide sequence of each member of the class may be readilydetermined by one skilled in the art by reference to the standardgenetic code, wherein different nucleotide triplets (codons) are knownto encode the same amino acid. An example of a polynucleotide encoding aYscF protein domain is depicted at SEQ ID NO:1. An example of apolynucleotide encoding a mature F1 protein domain is depicted at SEQ IDNO:3. An example of a polynucleotide encoding a LcrV protein domain isdepicted at SEQ ID NO:5. An example of a polynucleotide encoding afusion protein is depicted at SEQ ID NO: 7.

A fusion protein described herein that includes at least three proteindomains may include additional nucleotides flanking the coding regionencoding the fusion protein. The boundaries of a coding region aregenerally determined by a translation start codon at its 5′ end and atranslation stop codon at its 3′ end. In one embodiment, the additionalnucleotides include vector nucleotides. In another embodiment, theadditional nucleotides aid in expression of the fusion protein, such asexpression for subsequent isolation and optional purification.

A polynucleotide that encodes a fusion protein described herein can bepresent in a vector. A vector is a replicating polynucleotide, such as aplasmid, phage, or cosmid, to which another polynucleotide may beattached so as to bring about the replication of the attachedpolynucleotide. Construction of vectors containing a polynucleotidedescribed herein employs standard ligation techniques known in the art.See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual., ColdSpring Harbor Laboratory Press (1989). A vector can provide for furthercloning (amplification of the polynucleotide), e.g., a cloning vector,or for expression of the polynucleotide, e.g., an expression vector. Theterm vector includes, but is not limited to, plasmid vectors, viralvectors, cosmid vectors, and transposon vectors. A vector may bereplication-proficient or replication-deficient. A vector may result inintegration into a cell's genomic DNA.

Selection of a vector depends upon a variety of desired characteristicsin the resulting construct, such as a selection marker, vectorreplication rate, and the like. Suitable host cells for cloning orexpressing the vectors herein are prokaryotic or eukaryotic cells.Suitable eukaryotic cells include mammalian cells, such as yeast cells,murine cells, and human cells. Suitable prokaryotic cells includeeubacteria, such as gram-negative organisms, for example, E. coli.Suitable eukaryotic cells include, but are not limited to, humanembryonic kidney 293 (HEK293) cells.

An expression vector optionally includes regulatory sequences operablylinked to a polynucleotide encoding the fusion protein. An example of aregulatory sequence is a promoter. A promoter may be functional in ahost cell used, for instance, in the construction and/orcharacterization of a polynucleotide encoding a fusion protein describedherein, and/or may be functional in the ultimate recipient of thevector. A promoter may be inducible, repressible, or constitutive, andexamples of each type are known in the art. A polynucleotide encoding aprotein described herein may also include a transcription terminator.Suitable transcription terminators are known in the art.

A vector introduced into a host cell optionally includes one or moremarker sequences, which typically encode a molecule that inactivates orotherwise detects or is detected by a compound in the growth medium.Certain selectable markers may be used to confirm that the vector ispresent within the target cell. For example, the inclusion of a markersequence may render the transformed cell resistant to an antibiotic, orit may confer compound-specific metabolism on the transformed cell.Examples of a marker sequence include, but are not limited to, sequencesthat confer resistance to kanamycin, ampicillin, chloramphenicol,tetracycline, streptomycin, neomycin, puromycin, hygromycin, DHFR, GPT,zeocin, histidinol, and others.

In one embodiment, the vector is an adenoviral vector. Adenoviruses arenon-enveloped viruses 70-90 nm in diameter with an icosahedral capsid.Their genome is linear, double stranded DNA varying between 25-45kilobases in size with inverted terminal repeats (ITRs) at both terminiand a terminal protein attached to the 5′ ends (Russell, 2000, J GenVirol., 90:1-20). Their genome also encompasses an encapsidationsequence (Psi), early genes, and late genes. The principal early genesare contained in the regions E1, E2, E3 and E4. Of these, the genescontained in the E1 region are required for viral propagation. Theprincipal late genes are contained in the regions L1 to L5.

Adenoviruses have been used as the basis for a variety of vectors whichincorporate various coding regions. In each of these constructs, theadenovirus has been modified in such a way as to render it unable toreplicate following gene transfer. Thus, available constructs areadenoviruses in which genes of the early region, adenoviral E1, E2A,E2B, E3, E4, or combinations thereof, are deleted and into the sites ofwhich a DNA sequence encoding a desired protein can be inserted. Oneexample of an adenoviral vector routinely used is adenovirus serotype 5(Ad5). In the first Ad5 vectors, E1 and/or E3 regions were deletedenabling insertion of foreign DNA to the vectors (Danthinne andImperiale, 2000, Gene Ther., 7:1707-14; see also Rankii et al., U.S.Pat. No. 9,410,129, and Crouset et al., U.S. Pat. No. 6,261,807).Furthermore, deletions of other regions as well as further mutationshave provided extra properties to viral vectors. An example of anadenovirus encoding a fusion protein described herein is disclosed inClarke (US Patent Publication 2010/0209451). A viral vector, such as aadenoviral vector, can be present as a polynucleotide or as apolynucleotide inside a viral particle.

In one embodiment, a composition includes at least one fusion proteindescribed herein. In one embodiment, a composition includes a vectorencoding a fusion protein described herein. In one embodiment, thevector is an adenovirus vector, and the vector can be present in a viralparticle. Unless a specific level of sequence similarity and/or identityis expressly indicated herein (e.g., at least 80% sequence similarity,at least 90% sequence identity, etc.), reference to the amino acidsequence of an identified SEQ ID NO includes variants having the levelsof sequence similarity and/or the levels of sequence identity describedherein.

The compositions as described herein optionally further include apharmaceutically acceptable carrier. “Pharmaceutically acceptable”refers to a diluent, carrier, excipient, salt, etc., that is compatiblewith the other ingredients of the composition, and not deleterious tothe recipient thereof. Typically, the composition includes apharmaceutically acceptable carrier when the composition is used asdescribed herein. The compositions as described herein may be formulatedin pharmaceutical preparations in a variety of forms adapted to thechosen route of administration, including routes suitable forstimulating an immune response to an antigen. Thus, a composition asdescribed herein can be administered via known routes including, forexample, orally, parenterally including intradermal, transcutaneous andsubcutaneous, intramuscular, intravenous, intraperitoneal, etc., andtopically, such as, intranasal, intrapulmonary, intradermal,transcutaneous and rectally, etc. It is foreseen that a composition canbe administered to a mucosal surface, such as by administration to thenasal or respiratory mucosa (e.g., via a spray or aerosol), in order tostimulate mucosal immunity, such as production of secretory IgAantibodies, throughout the subject's body.

A composition described herein can be referred to as a vaccine. The term“vaccine” as used herein refers to a composition that, uponadministration to a subject, will increase the likelihood the recipientis protected against a Yersinia spp., such as Y. pestis.

A composition as described herein may be administered in an amountsufficient to treat certain conditions as described herein. The amountof fusion protein or vector present in a composition as described hereincan vary. In one embodiment, a dosage of viral particles containing avector that encodes a fusion protein described herein can be at least1×10⁸, at least 5×10⁸, at least 1×10⁹, or at least 5×10⁹ viralparticles, and no greater than 1×10¹⁰, no greater than 5×10¹⁰, nogreater than 1×10¹¹, or no greater than 5×10¹¹ viral particles. In oneembodiment, a dosage of a fusion protein (e.g., intramuscular) describedherein can be at least 0.01 micrograms (m), at least 0.1 μg, at least 1μg, or at least 10 μg, and no greater than 20 μg, no greater than 50 μg,or no greater than 100 μg.

The formulations may be conveniently presented in unit dosage form andmay be prepared by methods well known in the art of pharmacy. Methods ofpreparing a composition with a pharmaceutically acceptable carrierinclude the step of bringing the active compound (e.g., a viral particleor fusion protein as described herein) into association with a carrierthat constitutes one or more accessory ingredients. In general, theformulations are prepared by uniformly and intimately bringing theactive compound into association with a liquid carrier, a finely dividedsolid carrier, or both, and then, if necessary, shaping the product intothe desired formulations.

A composition can also include an adjuvant. An “adjuvant” refers to anagent that can act in a nonspecific manner to enhance an immune responseto a particular antigen, thus potentially reducing the quantity ofantigen necessary in any given immunizing composition, and/or thefrequency of injection necessary in order to generate an adequate immuneresponse to the antigen of interest. Adjuvants may include, for example,IL-1, IL-2, emulsifiers, muramyl dipeptides, dimethyl dioctadecylammonium bromide (DDA), avridine, aluminum hydroxide, magnesiumhydroxide, oils, saponins, alpha-tocopherol, polysaccharides, emulsifiedparaffins, ISA-70, RIBI, and other substances known in the art. It isexpected that proteins as described herein will have immunoregulatoryactivity and that such proteins may be used as adjuvants that directlyact as T cell and/or B cell activators or act on specific cell typesthat enhance the synthesis of various cytokines or activateintracellular signaling pathways. Such proteins are expected to augmentthe immune response to increase the protective index of the existingcomposition.

In another embodiment, a composition as described herein including apharmaceutically acceptable carrier can include a biological responsemodifier, such as, for example, IL-2, IL-4 and/or IL-6, TNF, IFN-α,IFN-γ, and other cytokines that effect immune cells. A composition canalso include other components known in the art such as an antibiotic, apreservative, an anti-oxidant, or a chelating agent.

Also provided are methods of using the compositions described herein.The methods include administering to a subject an effective amount of acomposition described herein. The subject can be, for instance, a human,a non-human primate (such as a cynomolgus macaque), a murine (such as amouse or a rat), a guinea pig, or a rabbit.

In some aspects, the methods may further include additionaladministrations (e.g., one or more booster administrations) of thecomposition to the subject to enhance or stimulate a secondary immuneresponse. A booster can be administered at a time after the firstadministration, for instance, one to eight weeks, such as two to fourweeks, after the first administration of the composition. Subsequentboosters can be administered one, two, three, four, or more timesannually. Without intending to be limited by theory, it is expected thatin some aspects annual boosters will not be necessary, as a subject willbe challenged in the field by exposure to microbes expressing proteinspresent in the compositions having epitopes that are identical to orstructurally related to epitopes present on proteins of the compositionadministered to the subject.

In one embodiment, a method includes an administration of a vector thatincludes a coding region encoding a fusion protein described herein. Thevector can be a viral vector, and the viral vector can be present in aviral particle. An example of a viral vector is an adenovirus. Theadministration of the vector can be topical, such as delivery to thenasal or respiratory mucosa. The administration of the vector can befollowed by a booster administration of an isolated or purified fusionprotein described herein. The booster can be parenteral, such asintramuscular, intradermal, or subcutaneous. Optionally, more than oneadministration of the vector can occur, and more than one administrationof the fusion protein can occur.

In one aspect, the invention is directed to methods for producing animmune response in the recipient subject. An immune response can behumoral, cellular, or a combination thereof. Antibody produced includesantibody that specifically binds the fusion protein. A cellular immuneresponse includes immune cells that are activated by the fusion protein.In this aspect, an “effective amount” is an amount effective to resultin the production of an immune response in the subject. Methods fordetermining whether a subject has produced antibodies that specificallybind a fusion protein, and determining the presence of a cellular immuneresponse, are routine and know in the art.

In one aspect the invention is also directed to conferring immunity toplague in a subject, including a human, caused by Yersinia spp., such asY. pestis. The plague can be pneumonic, bubonic, or septicemic.Conferring immunity is typically prophylactic—e.g., initiated before asubject is infected by a microbe causing plague, and is referred toherein as treatment of a subject that is “at risk” of infection. As usedherein, the term “at risk” refers to a subject that may or may notactually possess the described risk. Thus, typically, a subject “atrisk” of infection by a microbe causing plague is a subject present inan area where subjects have been identified as infected by the microbeand/or is likely to be exposed to the microbe even if the subject hasnot yet manifested any detectable indication of infection by the microbeand regardless of whether the subject may harbor a subclinical amount ofthe microbe. An example of a subject likely to be exposed to the microbeincludes a subject in the armed forces deployed at a location wherethere is risk of exposure to Y. pestis, such as a weaponized Y. pestis.While the methods described herein are of use in prophylactic treatment,the methods can also be used to treat a subject after the subject isinfected by the microbe. Accordingly, administration of a compositioncan be performed before, during, or after the subject has first contactwith the microbe, and the subject can have or be at risk of havingplague, such as pneumonic plague. Treatment initiated before thesubject's first contact with the microbe can result in increasedimmunity to infection by the microbe.

In another aspect, the method is directed to treating one or moresymptoms or clinical signs of certain conditions in a subject that canbe caused by infection by a microbe causing plague including Yersiniaspp., such as Y. pestis. As used herein, the term “symptom” refers tosubjective evidence of a disease or condition experienced by the patientand caused by infection by a microbe. As used herein, the term “clinicalsign” or, simply, “sign” refers to objective evidence of disease orcondition caused by infection by a microbe. The method includesadministering an effective amount of a composition described herein to asubject having a condition, or exhibiting symptoms and/or clinical signsof a condition, and determining whether at least one symptom and/orclinical sign of the condition is changed, preferably, reduced. Examplesof symptoms and/or clinical signs caused by a microbe causing plague,such as Y. pestis, are known to the person skilled in the art. Thesuccessful treatment of infection by Y. pestis in a subject is disclosedin Example 1, which demonstrates the protection against plague diseasecaused by Y. pestis in a mouse model and cynomolgus macaques byadministering a composition described herein. Mouse and cynomolgusmacaques models are a commonly accepted model for the study of diseasecaused by Y. pestis.

Also provided herein is a kit for immunizing a subject to protectagainst plague. The kit includes a vector described herein, such as anadenoviral vector, which includes a coding region encoding a fusionprotein described herein in a suitable packaging material in an amountsufficient for at least one administration. The kit also includes afusion protein described herein, in a suitable packaging material in anamount sufficient for at least one administration. Optionally, otherreagents such as buffers and solutions needed to administer the twocompositions are also included. Instructions for use of the packagedmaterials are also typically included. As used herein, the phrase“packaging material” refers to one or more physical structures used tohouse the contents of the kit. The packaging material is constructed bywell known methods, generally to provide a sterile, contaminant-freeenvironment. The packaging material may have a label which indicatesthat the materials can be used for conferring immunity to a subject. Inaddition, the packaging material contains instructions indicating howthe materials within the kit are employed to immunize a subject toprotect against plague. As used herein, the term “package” refers to acontainer such as glass, plastic, paper, foil, and the like, capable ofholding within fixed limits the materials and other optional reagents.“Instructions for use” typically include a tangible expressiondescribing the reagent concentration or at least one assay methodparameter, such as the relative amounts of reagent and sample to beadmixed, maintenance time periods for reagent/sample admixtures,temperature, buffer conditions, and the like.

The present invention is illustrated by the following example. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

Example 1

Currently, no plague vaccine exists in the United States for human use.The capsular antigen (Caf1 or F1) and two type 3 secretion system (T3SS)components, the low calcium response V antigen (LcrV) and the needleprotein YscF, represent protective antigens of Yersinia pestis. We useda replication-defective human type-5 adenovirus vector (Ad5) andconstructed recombinant monovalent and trivalent vaccines (rAd5-LcrV andrAd5-YFV) that expressed either the codon-optimized lcrV or the fusiongene YFV (made up of ycsF, caf1 and lcrV). Immunization of mice with thetrivalent rAd5-YFV vaccine by either the intramuscular (i.m.) or theintranasal (i.n.) route provided superior protection compared to themonovalent rAd5-LcrV vaccine against bubonic and pneumonic plague whenanimals were challenged with Y. pestis CO92. Pre-existing adenoviralimmunity did not diminish the protective response, and the protectionwas always higher when mice were administered one i.n. dose of thetrivalent vaccine (priming) followed by a single i.m. booster dose ofthe purified YFV antigen. Immunization of cynomolgus macaques with thetrivalent rAd5-YFV vaccine by the prime-boost strategy provided 100%protection to animals that had pre-existing adenoviral immunity, againsta stringent aerosol challenge dose of CO92. The vaccinated andchallenged macaques had no signs of disease, and the invading pathogenrapidly cleared with no histopathological lesions. This is the firstreport showing the efficacy of an adenovirus-vectored trivalent vaccineagainst pneumonic plague in mouse and NHP models.

INTRODUCTION

Yersinia pestis is the causative agent of plague, and can be transmittedto humans via an infected flea bite or by direct inhalation of theaerosolized bacilli from an infected person or an animal (1, 2). Plaguemanifests itself in three major forms in humans, namely bubonic,septicemic, and pneumonic (2). Pneumonic plague is the most feared formdue to its rapid onset and associated high mortality rate (1, 2). Y.pestis has been responsible for at least three pandemics in the past,which killed more than 200 million people (3). Current epidemiologicalrecords suggest 4,000 human plague cases annually worldwide (2) Theemergence of multi-antibiotic resistant Y. pestis strains from plaguepatients, and the potential of malicious dissemination of recombinantlyengineered bacteria as an airborne bioweapon, necessitates thedevelopment of an effective pre-exposure and/or post-exposureprophylaxis treatment (1, 2).

Currently, no Food and Drug Administration (FDA)-licensed plague vaccineexists in the United States, and recent efforts have focused on thedevelopment of recombinant subunit plague vaccines consisting of twowell-characterized Y. pestis antigens, the F1 capsular antigen, and thetype 3 secretion system (T3SS) component and effector LcrV (4-8). F1encoded by the caf1 gene has a polymeric structure and confers bacterialresistance to phagocytosis (9). The F1-V-based vaccines are generallyprotective against pneumonic plague in rodents and non-human primates(NHPs), and are currently undergoing clinic trails (10-17). However,considering the natural existence of fully virulent F1 minus Y. pestisstrains (18, 19) or those that have highly diverged LcrV variants (20,21), such F1-V-based vaccines would most likely not provide optimalprotection across all plague-causing Y. pestis strains in humans.

In an effort to search for new immunogenic antigens for the plaguesubunit vaccines, recent studies have shown that vaccination of micewith recombinant T3SS needle structure protein YscF (rYscF) providedprotection to mice against subcutaneous injection of the fully virulentand encapsulated Y. pestis strain CO92, and against an intravenouslyinjected pigmentation locus-negative Y. pestis KIM strain (22, 23).

In this study, we used a replication-defective human type-5 adenovirusvector (Ad5) to construct recombinant monovalent and trivalent(rAd5-LcrV and rAd5-YFV) vaccines that expressed either the lcrV or thefusion gene YFV (ycsF, caf1, and lcrV). We demonstrated the trivalentrAd5-YFV vaccine provided superior protection to immunized mice than themonovalent rAd5-LcrV vaccine against both bubonic and pneumonic plague,irrespective of whether or not the pre-existing adenoviral immunity wasartificially developed in these animals. Most importantly, one dose ofthe trivalent rAd5-YFV vaccine by the intranasal (i.n.) route inconjunction with a single dose of the purified recombinant fusionprotein rYFV by the intramuscular (i.m.) route in a prime-booststrategy, provided impressive (up to 100%) protection to both mice andcynomolgus macaques against high challenge doses of WT CO92 when givenby the aerosol route. Vaccinated NHPs rapidly cleared the pathogen withno signs of disease and histopathological lesions in various organs.

Materials and Methods

Bacterial strains and reagents. Y. pestis CO92 strain (WT CO92) wasisolated in 1992 from a fatal human pneumonic plague case and acquiredthrough the BEI Resources, Manassas, Va. The bioluminescent WT Y. pestisCO92 luc2 strain (WT CO92 luc2), which contains the luciferase operon(luc or lux), allowing in vivo imaging of mice for bacterialdissemination in real time, was previously constructed in our laboratory(26, 27). Y. pestis strains were grown in heart infusion broth (HIB)medium (Difco, Voigt Global Distribution Inc., Lawrence, Kans.) at 26 to28° C. with constant agitation (180 rpm) or on either 1.5% HIB agar or5% sheep blood agar (SBA) plates (Teknova, Hollister, Calif.). For theaerosol challenge, WT CO92 was grown in HIB enriched with 0.2% xylose(DL-xylose; Sigma-Aldrich, St. Louis, Mo.) as we previously described(28). Luria-Bertani (LB) medium was used for growing Escherichia coli at37° C. with agitation. Restriction endonucleases and T4 DNA ligase wereobtained from Promega (Madison, Wis.). Advantage cDNA PCR kits werepurchased from Clontech (Palo Alto, Calif.). All digested plasmid DNA orDNA fragments from agarose gels were purified using QIAquick kits(Qiagen, Inc., Valencia, Calif.).

Production and purification of recombinant proteins. Genes encodingYscF, Caf1 (F1), and LcrV were amplified from the genome of WT CO92 bypolymerase chain reaction (PCR) with the primer sets YscFHis_F.cln(CACATATGAGTAACTTCTCTGGATTTACGAAAG, SEQ ID NO:12) and YscFHis_R.cln(CACTCGAGTGGGAACTTCTGTAGGATGCCTT, SEQ ID NO:13), Caf1His_F.cln(CACATATGAAAAAAATCAGTTCCGTTATCG, SEQ ID NO:14) and Caf1His_R.cln(CACTCGAGTTGGTTAGATACGGTTACGGTTACAG, SEQ ID NO:15), LcrVHis_F.cln(CACATATGATTAGAGCCTACGAACAAAACCC, SEQ ID NO:16) and LcrVHis_R.cln(CAGTCGACTTTACCAGACGTGTCATCTAGCAGAC, SEQ ID NO:17), respectively. Theunderlines denote the restriction enzyme sites in the primers. Theamplified genes were individually cloned into the pET20b+ vector at theNdeI and XhoI restriction enzyme sites, which resulted in attaching ahistidine (His)-Tag at the C-terminus of each of the gene products. Inaddition, the yscF, caf1, and lcrV fusion gene (YFV) was syntheticallyconstructed by Epoch Biolabs, Inc. (Houston, Tex.) after codonoptimization for E. coli by using Blue Heron Biotechnology (Bothell,Wash.) online service (https://wwws.blueheronbio.com). A flexible linkerof 3× (GGGGS, SEQ ID NO:11) between YscF, Caf1 (F1), and LcrV domainswas added to facilitate correct folding of the fusion protein. Thefusion gene was similarly cloned into the pET20b+ vector with a His-Tagattached to the C-terminus of the YFV protein. Individual or the fusiongenes were expressed from E. coli BL21(DE3) (New England BioLabs,Ipswich, Mass.) after induction with 0.5 mM IPTG(isopropyl-beta-D-thiogalactopyranoside) for 4 h at 37° C. Therecombinant proteins (rYscF, rF1, rLcrV, and rYFV) were then purified byusing Ni²⁺-charged agarose (29). The recombinant F1 and LcrV fusionprotein (rF1-V) was purchased from the BEI Resources, and used as acontrol for some of the experiments.

Construction of recombinant adenoviruses. The lcrV and the YFV fusiongenes were codon optimized for expression in humans by using the BlueHeron Biotechnology online service, which also allowed us to optimizesecondary structures of the corresponding RNAs and removal of unwantedsites for the restriction enzymes, except for those used for cloningpurposes. The resulting constructs were designed to produce LcrV (37.2kDa), as well as the YFV fusion protein consisting of YscF (9.5 kDa),mature form of F1 (15.6 kDa), and LcrV (37.2 kDa), interconnected via aflexible linker, as mentioned above. To improve expression of thecorresponding genes, the Kozak consensus sequence was also placedupstream of the start codon. The constructs were then synthesized andverified via DNA sequence analysis by Epoch Biolabs, Inc. Each syntheticconstruct was cloned into pShuttleX vector (Clonetech Laboratories,Inc., Mountain View, Calif.) under the control of a CMV promoter.

To generate recombinant adenoviruses, the above gene constructs withtheir CMV promoters were removed from the pShuttleX vector and clonedinto the replication-defective human type-5 adenovirus plasmid vectorAdeno-X (Clonetech Laboratories, Inc.). The adenoviral constructs werecreated at the Baylor College of Medicine (BCM), Vector DevelopmentLaboratory, Houston, Tex. (available through the World Wide Web at theinternet site maintained by the Vector Development Laboratory, forinstance,bcm.edu/research/advanced-technology-core-labs/lab-listing/vector-development/adenovirus-vectors).The resulting recombinant plasmid vectors, Adeno-X/crV and Adeno-XYFVwere transfected separately into human embryonic kidney 293 (HEK293)cells and the plaque formation was monitored. After small-scaleexpansion, eight plaques from each of the recombinant vectortransfections were examined for the production of target proteins by dotblot analysis of the infected whole cell lysates with a monoclonalantibody to LcrV (mAb-LcrV) (BEI Resources). The positive plaques wereselected and designated as rAd5-LcrV and rAd5-YFV, respectively. Thecontrol adenovirus Ad5-CMV-Empty without recombinant gene insertion waspurchased from the BCM Vector Development Laboratory, and designated asAd5-empty.

The Ad5-empty, rAd5-LcrV, and the rAd5-YFV were then expanded on a largescale by using HEK293 cells in a chemically-defined, protein-free CD-293medium (Thermo Fisher Scientific, Waltham, Mass.) and purified at theBCM Vector Development Laboratory under GLP (good laboratory practice)conditions, and used for the subsequent studies. To examine expressionof the target protein-encoding genes in the stocked recombinant viruses,A549 human lung epithelial cells (American Type Culture Collection,Manassas, Va.) were infected with Ad5 constructs at 1000 viral particles(v.p.) per cell. The host cell lysates were harvested after 24 hpost-infection (p.i.). An aliquot of the cell lysates was then resolvedby SDS-PAGE and subjected to Western blot analysis with mAb-LcrVantibody. The purified rLcrV and rYFV antigens were used as controls. Asshown in FIG. 1, the size of the major band detected in the A549 celllysate infected with either the rAd5-LcrV (lane 2) or rAd5-YFV (lane 3)corresponded to the size of purified rLcrV (lane 5) or rYFV (lane 6). Noband was detected in the A549 cell lysate infected with the Ad5-empty(lane 4). The multiple bands detected in lanes 2, 3, and 6 most likelyrepresented degradation, or incomplete synthesis of the target proteins.

Animal studies. Six-to-eight-week old, female Swiss-Webster mice (17 to20 g) were purchased from Taconic Laboratories (Germantown, N.Y.). Allof the animal studies were performed in the Animal Biosafety Level(ABSL)-3 facility within the Galveston National Laboratory (GNL) underapproved Institutional Animal Care and Use Committee (IACUC) protocols.

1) Induction of Pre-Existing Immunity to Adenovirus in Mice.

To establish pre-existing immunity to adenovirus, animals received asingle dose of the Ad5-Empty by i.m. injection of 8×10⁹ v.p./100 μl intoboth quadriceps (50 μl each) 30 days prior to vaccination. Micereceiving saline (phosphate-buffered saline, PBS) served as a control.Blood was collected by the retro-orbital route before and 30 days afterthe Ad5-Empty injection, and microtiter plates pre-coated with 0.3μg/well of Ad5-empty were used to evaluate antibody titers toadenovirus. Animals with pre-existing adenovirus immunity weredesignated as PreAd-mice.

2) Immunization of Mice with the Recombinant Proteins or Recombinant Ad5Constructs.

Naïve mice (40 per group) were immunized with either the mixture ofthree recombinant proteins (rYscF, rF1, and rLcrV, 25 μg/each) or 45 μgof the corresponding recombinant fusion protein (rYFV) via the i.m.route. The antigens were emulsified 1:1 in Imject Alum adjuvant (PierceCompanies, Dallas, Tex.). One primary immunization and two identicalboosters were given on days 0, 15 and 30. Naïve mice receiving adjuvantalone served as a control. For the recombinant Ad5 constructs, naïvemice or preAd-mice (40 per group) were either i.m. or i.n. immunizedwith one dose (8×10⁹ v.p) of rAd5-LcrV monovalent or rAd5-YFV trivalentvaccine. Control animals (both naïve and preAd-mice) received the samedose of Ad5-empty via the same route as their corresponding immunizedmice. In some cases, the dose of Ad5-Empty was split equally into i.m.injection and i.n. instillation for the control naïve mice. During i.m.immunizations, the dose in a 100 μl volume was equally split andinjected into both quadriceps, while for the i.n. immunizations, thedose in 40 μl was equally distributed into each of the nares of micefollowed by 20 μl of PBS wash.

3) Immunization of Mice with the Combination of rAd5-YFV and rYFV.

PreAd-mice (20 per group) were first i.n. immunized with 8×10⁹ v.p./40μl of rAd5-YFV trivalent vaccine and then followed (two weeks later) byi.m. immunization with 10 μg rYFV (emulsified 1:1 in Alum adjuvant).PreAd-mice immunized with either 10 μg of rYFV or 8×10⁹ v.p./40 μl ofrAd5-YFV alone were used for comparison, and PreAd-mice without furtherimmunizations served as a negative control.

4) Evaluation of Antibody Titers in Mice.

Blood was collected by the retro-orbital route from all vaccinated andcontrol mice at day 0 and after 12-15 days of last vaccination. Serawere separated and the antigen-specific antibodies were then evaluated.Briefly, ELISA plates were pre-coated with 200 ng/well of therecombinant proteins (e.g., rLcrV, rF1 or rYscF). Two-fold seriallydiluted sera was then added in the wells of the ELISA microtiter plates,followed by the addition of secondary horseradish peroxidase(HRP)-conjugated anti-mouse specific antibodies to IgG, its isotypes,and/or IgA. The ELISA was performed as we described previously (30).

5) T-Cell Responses.

T cells were isolated from splenocytes of PreAd-mice (n=5) immunizedwith either rAd5-YFV (i.n., 8×10⁹ v.p) alone or in a prime-boostcombination with rYFV (10 i.m.) on day 15 after the last immunization.The isolated T cells were co-cultured with γ-irradiated splenocytes fromnaïve mice (severed as antigen-presenting cells [APCs]) pulsed orun-pulsed with F1-V fusion protein, 100 μg/ml. After 72 h of incubation,1 μCi of [³H] thymidine was added into each well, and the cellsharvested 16 h later using a semi-automated sample harvester, FilterMateHarvester (PerkinElmer, Waltham, Mass.), followed by the measurement ofradioactive counts (TopCount NXT, PerkinElmer) as we previouslydescribed (31, 32). To measure interferon (IFN)-γ producing T cells, theisolated T cells were incubated with the pulsed and un-pulsed APCs for 2days and evaluated by the enzyme-linked immunospot (Elispot) assay (R&DSystems Inc., Minneapolis, Minn.).

6) Challenge and Re-Challenge.

Mice were challenged with WT CO92 on day 14-15 post last vaccination byeither the subcutaneous (s.c.), i.n., or the aerosol route as wepreviously described (28, 33). Aerosolization was performed using a6-jet Collison nebulizer attached to a whole-body mouse aerosol chamber.The challenge doses ranged from 24 to 8,500 LD₅₀ for the s.c. route and21 to 800 LD₅₀ for the i.n. route. The presented dose (Dp) for theaerosol challenge was calculated to be in the range of 3.14 to 6.34×10⁵colony forming units (CFU). The LD₅₀ of WT CO92 for Swiss-Webster miceis ˜50 CFU for developing bubonic plague (s.c.), ˜500 CFU for inducingpneumonic plague (i.n.), and ˜Dp of 2100 CFU for the aerosol route (28,32). For the re-challenge experiment(s), on day 32 after the initial WTCO92 aerosol challenge, the vaccinated mice that survived were infectedi.n. with 100 LD₅₀ of the WT CO92 luc2 strain. The age matched naïvemice served as a control. The animals were imaged on day 3 p.i. with WTCO92 luc2 strain by using an in vivo imaging system (IVIS) 200bioluminescent and fluorescence whole-body imaging workstation (CaliperCorp. Alameda, Calif.) in the ABSL-3 facility.

Non-human primate (NHPs) study. Cynomolgus macaques (2.5-3.5 kg, males)were purchased from Prelabs, Hines, Ill. The NHPs were sedated by theadministration of ketamine i.m. during the procedures, and all of thestudies were performed in the ABSL-3 facility under an approved IACUCprotocol.

1) Induction of Pre-Existing Immunity to Adenovirus and Immunization.

To induce pre-existing immunity, four randomly selected NHPs wereinjected in the left quadriceps muscle with 5×10¹⁰ v.p./250 μl ofAd5-Empty (day 0). After 30 days, these NHPs were i.n. immunized with1×10¹¹ v.p./500 μl of rAd5-YFV, followed by 50 μg/250 μl of rYFV boost(emulsified 1:1 in Alum adjuvant) via the i.m. route on day 42. In thecontrol group, four NHPs received 250-500 μl of saline at days 0, 30 and42 via the same routes as the immunized NHPs, and served as controls(Table 1). The nasal administration of rAd5-YFV was performed by using aMucosal Atomization Device (MAD Nasal, Wolfe Tory Medical, Inc., SaltLake City, Utah) that delivers intranasal medication in a fine mist,thus enhancing the absorption and improving bioavailability.

TABLE 1 NHP immunization and challenge timeline Induction of preexistingGroup anti-adenovirus immunity Prime vaccination Boost with rYFV AerosolChallenge (size) (Day 0) (Day 30) (Day 42) (Day 85) Immunized 5 × 10¹⁰v.p./250 μl Ad5- 1 × 10¹¹ v.p./500 μl 50 μg of the rYFV mixed (4) emptyi.m. route rAd5-YFV i.n. with alhydrogel (250 μl) route (250 μl pergiven by the i.m. route nostril) WT CO92 (Dp: 1.32 to 8.08 × 10⁷ CFU)Control Saline (250 μl) i.m. route Saline (500 μl) i.n. Saline (250 μl)i.m. route (4) route (250 μl per nostril)

2) Aerosol Challenge.

The immunized and control NHPs were challenged with WT CO92 by theaerosol route on day 85 (Table 1). Briefly, WT CO92 was aerosolized witha 6-jet Collison nebulizer. The nebulizer was attached to a head-onlyNHP aerosol exposure box and real-time plethysmography was performed oneach of the anesthetized NHP during aerosol challenge. Theaerosol/plethysmography system was controlled by a Biaera AeroMP aerosolplatform (Biaera Technologies, LLC Hagerstown, Md.) integrated with arespiratory inductive plethysmography (RIP) system (Data SciencesInternational St. Paul, Minn.). Aerosol samples were collected duringeach animal exposure by using all glass BioSamplers to assure accurateaerosol delivery, and the corresponding Dps were calculated (28, 34).

The NHPs were monitored and evaluated closely for developing clinicalsigns of the disease. Clinical scores were provided after thoroughexamination of the animals by the veterinarian staff. The NHPs wereeuthanized when they were found with a clinical score of 8 and above.The parameters examined but not limited to included absence of grooming,decreased breathing, and non-responsive to human presence at cage side.All NHP exposures to aerosols of WT CO92 were performed in our ABSL-3facility within the GNL in a specialized aerobiology suite equipped witha Class III biosafety glove cabinet.

3) Antibody Titers, Blood Cell Counts, and Bacterial Burden.

Blood samples were collected from the femoral veins of NHPs at varioustime points during the experiment. Antibody titers to Ad5, LcrV, F1, andYscF on days 0, 42, 56, 85, 98 and 112 were evaluated by ELISA as wedescribed above. The last two time points (days 98 and 112) correspondedto days 14 and 28 after WT CO92 challenge. Blood cell counts wereanalyzed on the day of WT CO92 challenge (day 85) and on days 3 and 6post challenge by using a Drew Scientific Hemavet 950 hematology system(Drew Scientific, Inc., Dallas, Tex.). The bacterial loads wereevaluated by plating the blood samples which were drawn when controlNHPs were euthanized (on day 3 or 4 post WT CO92 challenge) or atvarious time points (e.g., days 3, 6, 14, 28, 70, and 82) post WT CO92challenge in the case of immunized NHPs.

4) Necropsy and Histopathological Analysis.

After euthanasia, necropsies were performed by the certified chiefbiocontainment veterinarian at UTMB. NHP organs, such as lungs, liver,spleen, and the lymph nodes (hilar, submandibular, and mediastinal) wereremoved and grossly examined. A portion of these organs was homogenizedand plated for assessing bacterial load (35), while another portion wasfixed in 10% neutral buffered formalin (33, 36) and tissues processedand sectioned at 5 μm. The samples were mounted on slides and stainedwith hematoxylin and eosin (H&E). Sections from the lungs were alsosubjected to Gram stain to examine the presence of plague bacilli.Tissue lesions were scored on the basis of a severity scale, whichcorrelated with estimates of lesion distribution and the extent oftissue involvement (minimal, 2 to 10%; mild, >10 to 20%; moderate, >20to 50%; severe, >50%), as previously described (33, 36). Thehistopathological evaluation of the tissue sections was performed in ablinded fashion.

CT scans. CereTom NL 3000 (Neurologica, MA), which is an eight-slicetomography with high-contrast resolution of 0.6 mm (developed for humanhead imaging in ICU), was used. The image acquisition settings were:tube voltage, 100 kV; tube current, 5 mA; and axial mode with slicethickness of 1.25 mm. Image resolution was 512×512 pixels. The imagesharpness was optimized to soft tissue.

Statistical analysis. Two-way analysis of variance (ANOVA) with theTukey's post hoc test or the multiple Student's t-test with theHolm-sidak post hoc test correction was used for data analysis. We usedKaplan-Meier survival estimates for animal studies, and p values of≦0.05 were considered significant for all of the statistical tests used.

Results

Immunogenicity of rYFV fusion protein. Mice were i.m. immunized witheither the mixture of recombinant proteins (rYscF+rF1+rLcrV) or thefusion protein rYFV. Both recombinant proteins (rYFV or rYscF+rF1+rLcrV)conferred 100% protection to mice when challenged by either the s.c.route (8500 LD₅₀, to induce bubonic plague) or the i.n. route (800 LD₅₀,to induce pneumonic plague) with WT CO92, while developing significantantibody titers to LcrV (FIG. 2).

Protective immunity of the recombinant adenoviruses in both bubonic andpneumonic plague mouse models. Mice were immunized i.m. or i.n. withrAd5-LcrV monovalent or rAd5-YFV trivalent vaccines to evaluate theirpotential to protect animals from plague. Irrespective of theimmunization route, mice that were administered rAd5-YFV trivalentvaccine displayed 100% protection when challenged with 60 LD₅₀ of WTCO92 in a bubonic plague model (FIG. 3A) However, only 50 to 55% of micereceiving the rAd5-LcrV monovalent vaccine were protected and allcontrol mice died by day 11 p.i. (FIG. 3A). In a more stringentpneumonic plague model (90 LD₅₀ of WT CO92), animals vaccinated by thei.n. route with rAd5-YFV trivalent vaccine were 60% protected, while thesurvival rate declined to 10% when immunization occurred by the i.m.route (FIG. 3B). In comparison, either none or 20% of the animalsimmunized with the Ad5-LcrV monovalent vaccine survived when vaccinationoccurred by i.m. versus the i.n. route. Overall, these data indicatedvaccines to be more effective when instilled by the i.n. route. Thecorresponding control mice (receiving Ad5-empty by the i.m. or the i.n.route) succumbed to infection by day 4 p.i. (FIG. 3B).

Higher antibody titers to LcrV were generally observed in mice thatreceived the rAd5-YFV trivalent vaccine when compared to that of therAd5-LcrV monovalent vaccine-immunized animals, reaching statisticallysignificant levels for IgG1 in mice that were immunized by the i.n.route (FIG. 3C). In terms of immunization routes, i.n. vaccinated miceoverall had superior antibody titers when compared to animals immunizedby the i.m. route, reaching statistical significance for the productionof IgG1 and IgA (FIG. 3C). Irrespective of the recombinant virus usedand route of immunization, all of the vaccinated mice developed a morebalanced Th1 and Th2 type antibody responses when compared toimmunization of animals with the recombinant proteins (FIGS. 2A and 3C).

Pre-existing immunity to adenovirus in mice. The adenoviral antibodytiters on day 30 after injection of Ad5-empty in mice ranged from102,400 to 819,200. In a bubonic plague model, at a 24 LD₅₀ challengedose, a similar level of protection (80 to 90%) was noted in miceimmunized with rAd5-YFV trivalent vaccine, irrespective of whether ornot pre-existing antibodies to adenovirus were developed (FIG. 4A). Incontrast, the survival rate was 40% in mice without pre-existing Ad5antibodies and only 10% in preAd-mice when immunization occurred withthe rAd5-LcrV monovalent vaccine (FIG. 4A). In a pneumonic plague model(21 LD₅₀), rAd5-YFV-immunized mice with or without pre-existing immunityto Ad5 exhibited a similar 55-60% survival rate which was much higherthan in mice immunized with the rAd5-LcrV monovalent vaccine with orwithout pre-immunity to Ad5 (10-20% protection) (FIG. 4B). All of thecontrol mice died on the indicated days in a bubonic or pneumonic plaguemodel (FIGS. 4A and 4B).

Balanced Th1 and Th2 type antibody responses with robust titers to LcrVwere observed across all immunized mice (FIG. 4C). However, twoimportant observations were drawn from this study: 1) compared torAd5-LcrV monovalent vaccine immunized mice, animals that werevaccinated with the rAd5-YFV trivalent vaccine generally developedbetter antibody titers (both IgG and its isotypes as well as IgA) toLcrV, although some did not reach statistical significance (e.g., IgG1and IgG2a in preAd-mice as well as IgA), and 2) mice withoutpre-existing adenoviral immunity developed slightly higher IgG and IgAantibody titers to LcrV compared to that of preAd-mice receiving thetrivalent rAd5-YFV vaccination, although only total IgG and its isotopesreached statistical significance (FIG. 4C). As expected, none of theunimmunized control mice developed any detectable level of protectiveanti-LcrV antibodies, and, thus, succumbed to infection (FIGS. 4A and4B). Importantly, in spite of slight lower antibody titers to LcrV inmice with pre-existing Ad5 antibodies, animals were similarly protectedwhen the Ad5-YFV trivalent vaccine was administered by the i.n. routeagainst challenge with WT CO92 in both bubonic and pneumonic plaguemodels (FIGS. 4A and 4B).

Prime-boost and aerosol challenge. Our above data indicated that thetrivalent rAd5-YFV vaccine was better than the monovalent rAd5-LcrVvaccine in providing protection to mice against Y. pestis infection.However, the overall protection rate did not reach 100% in the pneumonicplague model (FIGS. 3B and 4B). To enhance protection, a boost with rYFV(10 μs) was administered to mice i.m. two weeks later following i.n.instillation of the rAd5-YFV trivalent vaccine. As shown in FIG. 5, miceimmunized with only rAd5-YFV had a 70% survival rate after aerosolexposure of WT CO92, irrespective of whether or not pre-existingadenoviral immunity was developed. The preAd-mice vaccinated with thecombination of rAd5-YFV and rYFV displayed a protection rate of 80% withan overall delayed death pattern after WT CO92 aerosol challenge at a Dpof 6.34×10⁵ CFU (˜302 LD₅₀). The rYFV-immunized mice alone (single dose,no boosts) had 5% survival, and all unimmunized preAd-mice died afteraerosol exposure of the pathogen between days 3 to 5 p.i. (FIG. 5).

To further evaluate the potential of the prime-boost strategy, anotherset of immunized mice were exposed to a slightly lower WT CO92 aerosolchallenge dose (Dp of 4.62×10⁵ CFU, ˜220 LD₅₀). As shown in FIG. 6A, thepreAd-mice first vaccinated with the rAd5-YFV trivalent vaccine and thenboosted with rYFV, were 100% protected against developing pneumonicplague. On the other hand, preAd-mice that were vaccinated with only therAd5-YFV trivalent vaccine showed 55% survival rate, with all theunimmunized preAd-mice succumbed to infection by day 3 post challenge.

In addition, 55-60% of T cells isolated from the prime-boost group ofmice were IFN-γ positive, while this number was only 30% for mice thatwere immunized with rAd5-YFV trivalent vaccine alone (FIG. 6B). However,there was no difference between the two immunized groups of mice (withor without the prime-boost) in terms of their T cell proliferativeresponses upon stimulation with the F1-V antigen (FIG. 6C).

In terms of antibody production, we noted that IgG, its isotypes, andIgA antibody titers to the three antigens (F1, LcrV, and YscF) weregenerally higher in the prime-boost group of mice over those animalsthat only received the rAd5-YFV trivalent vaccine. Further, a balancedTh1 and Th2-based antibody responses were observed (FIG. 7A-7C).

Continued protection of mice conferred by prime-boost vaccinationstrategy against the initial aerosol and then the subsequent intranasalWT CO92 challenge. In our subsequent experiment, preAd-mice werevaccinated with either the rAd5-YFV trivalent vaccine alone or with arYFV boost. The preAd-mice receiving the Ad5 empty vector alone servedas a control. After the vaccination regimen, mice were subjected to WTCO92 aerosol challenge with still a relatively lower Dp (3.14×10⁵ CFU,˜150 LD₅₀) as compared to the above two aerosol challenges (FIGS. 5 and6A). As noted in FIG. 8A, 100% of the animals survived the initialchallenge in all of the immunized groups, while 90% of the control micedied (FIG. 8A). After 32 days of the initial aerosol challenge, thesurvivals from the immunized groups were re-challenged with 100 LD₅₀ ofWT CO92 luc2 strain by the i.n. route, and the age-matched uninfectednaïve mice (n=5) served as a control. As shown in FIG. 8A, 80% of themice were protected from developing plague in the rAd5-YFV-immunizedgroup, while this protection was 100% when the prime-boost strategy wasused. In contrast, all of the naïve re-challenge control mice succumbedto infection within day 4 p.i. The bioluminescent images showed that theplague bacilli disseminated from the initial infection site of lungs tothe whole body in all 5 naïve control mice after day 3 p.i. (FIG. 8B-I).On the other hand, no animals were positive in the group that receivedvaccination by the prime-boost regimen (FIG. 8B-II). However, one mousefrom the rAd5-YFV-immunized group was bioluminescent positive, with theorganisms confined in the lungs (FIG. 8B-III). Thisbioluminescent-positive animal along with another one mouse in the samegroup, which did not show bioluminescence at the time of imaging (day 3p.i.), eventually died, resulting in the overall survival rate of 80% inthe rAd5-YFV immunized group of mice (FIG. 8A).

Evaluation of protection provided by the trivalent rAd5-YFV vaccine incynomolgus macaques against aerosol challenge of WT CO92. Four NHPs wereinitially i.m. injected with Ad5-empty to generate pre-existingadenoviral immunity. This was followed by one dose of the rAd5-YFV bythe i.n. instillation in the form of mist, and then one dose of the rYFVby the i.m. route. Four unimmunized NHPs served as a control (Table 1).These NHPs were then challenged with the aerosolized WT CO92 at Dpranging from 1.32 to 8.08×10⁷ CFU (˜13,200-80,800 LD₉₀, with 1 LD₉₀=864CFU (37)). No clinic signs were noted in the immunized group of NHPs,and the animals remained healthy and survived the WT CO92 challengeuntil euthanized at the end of the study (FIG. 9). The CT scans ofimmunized NHPs, that survived the WT CO92 challenge (FIG. 9) andeuthanized on day 82 post challenge, did not display any abnormalitiesin the lungs and their surrounding areas when compared to the images ofthe animals before the WT CO92 challenge on day 85 (FIG. 10) (Table 1).In contrast, the control NHPs euthanized on day 3-to-4 post challenge,showed consolidation in both the right and the left lung, an indicationof severe inflammation (FIG. 10).

Necropsy on immunized NHPs was performed 82 days after the WT CO92challenge, and no gross abnormities were observed, and the internalorgans (lungs, liver, spleen and the lymph nodes) were all free ofbacteria (Table 2). In contrast, all unimmunized control NHPs developedclinical signs of the disease as early as 36 h p.i. and reached aclinical score of 8 and higher on day 3-to-4 p.i. The control NHPs hadcough, abnormal respiration, lethargy, and a hunched posture. Althoughwe did not notice fever in these animals during the progression of thedisease, it could be related to not continuously monitoring these NHPsby using telemetry. Necropsy of these animals revealed seroushemorrhagic fluid in the thorax with respiratory frothy serousdischarge. Lungs were hyper-inflated with hemorrhagic frothy fluid, andthe spleen, liver and the lymph nodes were enlarged. The highestbacterial loads (1.12 to 1.26×10⁹ CFU/node) were noted in the hilarlymph nodes and lungs (2.22×10⁷ to 1.06×10⁹ CFU/g) followed by the liver(8.16×10⁶ to 1.69×10⁷ CFU/g), spleen (2.13 to 4.47×10⁶ CFU/g) andsubmandibular lymph nodes (2.33×10⁵ CFU/node). Only one animal showedbacteria in the blood with a count of 2500 CFU/ml, and no bacilli wasdetected in the other control NHPs (Table 2).

TABLE 2 NHP clinical score, bacterial loads and necropsy report DaysPost Clinical NHP Infection Bacterial loads in blood/organs ScoreNecropsy Report Control 3-4 Blood: 0-2500 CFU/ml ≧8 External: Thin,pale, dehydrated and Lung: 2.22 × 10⁷-1.06 × 10⁹ CFU/g scruffy coatLiver: 8.16 × 10⁶-1.69 × 10⁷ CFU/g Respiratory: Frothy serous discharge;Spleen: 2.13-4.47 × 10⁶ CFU/g hyper-inflated with hemorrhagic Hilarlymph node: 1.12-1.26 × 10⁹ frothy fluid (~50 mL) CFU/node Lymphatic:Enlarged submandibular node Submandibular lymph node: Spleen: Firm andenlarged 2.0-2.33 × 10⁵ CFU/node Liver: Firm, enlarged and rounded edgesLocomotion: Lethargic Body Cavities: Serous hemorrhagic fluid in thorax(~50 mL) Immunized 82 Negative for all the organs; blood   0 Normalsamples were negative for bacteria as early as day 3 post infection

NHP blood cell counts and antibody titers. The changes in the blood cellcounts in immunized NHPs versus the control after WT CO92 challenge areshown in FIG. 11. Only the lymphocyte (LY) counts in the control NHPsfell below the normal range by day 3 post WT CO92 challenge before theywere euthanized. However, in the immunized NHPs, LY counts remainedwithin the normal range on days 3 and 6 post WT CO92 challenge.

Both immunized and control NHPs showed some level of pre-existing Ad5antibody titers (6,400-25,600) on day 0 as a consequence of naturallyacquired infection with adenoviruses. The anti-Ad5 titer was increasedto 409,600 on day 30 in immunized NHPs after receiving the rAd5-Emptyinjection, and continued to climb up slightly on days 42 and 56 as aresult of immunization with rAd5. The anti-Ad5 antibody titer wasmaintained at a similar level to that observed on day 0 in the controlNHPs (FIG. 12A). No pre-existing anti-LcrV, anti-F1, and anti-YscFantibodies were detected in both the groups of NHPs before immunization(data not shown). However, high antibody titers to three Y.pestis-specific antigens (e.g., F1, LcrV, and YscF) were noticed in allof the immunized NHPs (FIG. 12B-12E). Compared to the antibody titers onday 42, the antigen specific IgG antibodies increased ˜10 fold for LcrVand YscF, but nearly 1000 fold for F1 on day 56 (FIG. 12B-12D). Thus,boost on day 30 with rYFV (Table 1) led to increase in antibody titers.These antigen-specific antibody titers slightly decreased on day 85 (theday of challenge). A similar trend was observed for the anti-LcrV IgAantibody titers, which were increased ˜10 fold on day 56 after the rYFVboost (FIG. 12E). Compared to all three antigen-specific IgG antibodytiters, the anti-LcrV titers were the highest followed by anti-YscF andanti-F1 across the course of immunization, and the difference couldreach up to 1000 fold (anti-LcrV vs anti-F1 on day 42) (FIGS. 12B and12C). After WT CO92 aerosol challenge, anti-F1 IgG titers were furtherboosted, while sustaining IgG titers for LcrV and YscF, and IgA LcrVtiters up to 28 days post WT CO92 challenge (overall day 112 afterinitiation of vaccination) (FIG. 13A-13E).

NHP histopathological analysis. As shown in FIG. 14, the unimmunizedcontrol NHPs showed marked acute inflammatory reactions in the lungs,pleura, and the mediastinal lymph nodes. Specifically, multifocalhemorrhage and diffused supportive inflammation were observed in thelungs with no alveolar spaces. Similar changes were also observed inpleura and mediastinal lymph nodes of these unimmunized NHPs.Furthermore, tissue sections from the lungs with Gram staining revealedthe presence of bacteria, presumptively Y. pestis (FIG. 14, inset).Interestingly, the liver and the spleen tissues of unimmunized NHPsshowed normal morphological characteristics in spite of higher bacterialloads (Table 2), indicating that pneumonic changes are the primary causeof death in control groups. In the immunized NHP group, the lungs,pleura, mediastinal lymph nodes, and the liver were normal, and thelungs had alveolar spaces. The only notable and expected changesobserved in the prime-boost group was the hyperplasia of lymphoidfollicles in mediastinal lymph nodes and the spleen. These changes canmainly be attributed to reaction of vaccination.

DISCUSSION

Historically, vaccination has not only been one of the most significantadvances in healthcare, but also a cost-effective means of public healthintervention. The high mortality rate associated with pneumonic plague,the potential use of Y. pestis as a biological weapon, and the currentlack of a FDA approved plague vaccine highlight the importance of ourstudies.

Previously, the plague vaccine licensed in the U.S. (sold under the nameof USP) was a formaldehyde-killed preparation of the highly virulent195/P strain of Y. pestis; however, the production of this vaccine wasdiscontinued in 1999. The vaccination regimen included a course ofinjections over a period of 6 months, and then the annual boosters (38,39). The vaccine was effective against bubonic plague, but protectionagainst pneumonic plague was uncertain. The incidence of side effects,such as malaise, headaches, elevated body temperature, andlymphadenopathy was high; and the vaccine was expensive (40). Alive-attenuated vaccine based on Y. pestis pigmentation locus negativeEV76 strains is also available in some parts of the world where plagueis endemic (1). These types of vaccines have existed since the firsthalf of the 20^(th) century and have proven effective against bothsubcutaneous and inhalation challenges with Y. pestis. However, theEV76-based vaccines are not genetically uniform and are also highlyreactogenic (41), and, hence, would not meet the standards for FDAapproval.

The major problems encountered in developing live-attenuated vaccinesare inadequate attenuation, particularly in immunocompromisedindividuals, and the potential to revert back to the virulent phenotype.Efforts have been made to generate well-characterized andrationally-designed attenuated plague vaccines. For example, mutationsthat effectively attenuate Salmonella such as aroA, phoP, htrA and lppgenes, were introduced in Y. pestis, but these mutations had only alimited effect on Y. pestis virulence (33, 42-44). Similarly, a deletionof the Y. pestis global regulator gene rovA, significantly attenuatedthe bacterium during subcutaneous infection, but this mutant was onlyslightly attenuated when given via an intranasal or the intraperitonealroute (45). Recently, a highly attenuated Δlpp ΔmsbB Δail triple mutant,which was deleted for genes encoding Braun lipoprotein (Lpp), anacetyltransferase (MsbB), and the Attachment Invasion Locus (Ail), wasconstructed (27). Mice immunized with this triple mutant via either theintranasal, subcutaneous, or the intramuscular route, were protectedfrom lethal WT CO92 challenge, and thus could be an excellent vaccinecandidate (27, 35). This triple mutant was subsequently excluded fromthe CDC select agent list in May 2016. However, further evaluation ofthe efficacy of this triple mutant in higher animal models is warranted.

While the above conventional vaccine strategies have focused onlive-attenuated or killed bacterial approaches, a new method in thedevelopment of vaccines uses platform technologies to overcome some ofthe challenges in vaccine design. The adenoviral vector system has beensuccessfully used as a vaccine platform for a number of pathogens,including Y. pestis (46, 47), with several advantages: 1) the adenoviralgenome is well characterized with the capability of integrating ≧6-kb ofthe potential insert size for delivering multiple antigens; 2) thereplication-defective Ad5 vector has been developed for gene therapeuticapplications at a wide range of doses, with minimal side effects; and 3)adenoviruses have a broad tropism infecting a variety of dividing andnon-dividing cells. Studies have shown that adenoviruses transfer geneseffectively to APCs in vivo to promote rapid and robust humoral andcellular immune responses to the transgene products (48-55). Inaddition, adenoviruses can be grown to high titers in tissue culturecells and can be applied systemically as well as through mucosalsurfaces, and are relative thermostable to facilitate their clinicaluse.

Our rAd5-YFV trivalent vaccine had an average yield of 1×10¹⁶ v.p. perbatch in a cell suspension culture in CD 293 Medium. The vaccine wasfree of proteins, serum, and animal-derived components, thus making itsuitable for a broad range of prophylactic and therapeutic use. Comparedto a favored Th2 response in mice immunized with rYFV or a mixture ofrYscF, rLcrV, and rF1 (given with alum which skews the immune responseto Th2) (FIG. 2A), a more balanced Th1- and Th2-based antibody responsewas observed in mice immunized with the rAd5 vaccines (FIGS. 3C, 4C, and7A-7C). Indeed, Ad5 has been shown to promote Th1 response (47). Asexpected, intranasal administration of rAd5-LcrV monovalent and rAd5-YFVtrivalent vaccines elicited IgA production in immunized animals (bothmice and NHPs), and most importantly, mice immunized with rAd5-YFV aloneor in a prime-boost vaccination strategy, exhibited a robust T cellproliferative responses (FIG. 6C). These features suggest superiority ofAd5-based vaccines over the rF1-V-based subunit vaccines, as theprotection of the latter vaccines is largely dependent on systemicantibody responses without mucosal and cellular immune components.Interestingly, although generally a higher IgG antibody titer wasobserved across all mice immunized intranasally when compared to animalsimmunized intramuscularly with the recombinant adenoviruses, theprotection rate was indistinguishable during the development of bubonicplague. However, subtle differences in protection were noted dependingupon of the route of immunization of mice in a pneumonic plague model(FIGS. 3A and 3B), which further highlighted the importance of mucosalimmunity during the development of pneumonic plague.

Pneumonic plague begins with an anti-inflammatory state (i.e., first 24to 36 h after infection), which is characterized by a delay in theinflammatory cell recruitment to the lungs and production ofpro-inflammatory cytokines and chemokines (56). Therefore, a plaguevaccine should be able to stimulate a strong mucosal immunity toovercome this initial immune suppression in the host (57). In our futurestudies, we plan to discern the role of mucosal immune response (e.g.,IgA) that is triggered by the rAd5-YFV vaccine in protection.

Compared to the monovalent rAd5-LcrV vaccine, the trivalent rAd5-YFVvaccine not only mounted higher anti-LcrV antibody titers (both IgG andIgA) (FIGS. 3C and 4C) but also generated immune responses to the F1 andYscF (FIG. 7), which correlated with better protection of animalsagainst both bubonic and pneumonic plague (FIGS. 3A and 3B, 4A and 4B,and 5). In addition, LcrV was more immunogenic than F1 and YscF in bothmice and NHPs that were immunized with the trivalent rAd5-YFV vaccine(FIGS. 7 and 12). In contrast, the antibody titers to F1 were the lowestamong the three examined antigens in the rAd5-YFV-immunized NHPs (FIG.12). The difference in immunogenicity may be attributed to the nature ofeach of the antigens; however, conformation of the antigens in thefusion protein may also play a role, especially as higher anti-LcrVantibody titers were observed in the rAd5-YFV-immunized mice than inrAd5-LcrV vaccinated animals. Alternatively, the presence of other twoantigens could augment antibody production to LcrV.

Previously, a rAd5 (designated as rAdsecV) expressing a human Igksecretion (sec) signal fused to lcrV was reported (46). The rAdsecVproduced a secreted form of LcrV and elicited specific T cell responsesas well as high IgG titers in sera, which protected mice from a lethalintranasal challenge of Y. pestis CO92 in a single intramuscularimmunization (46). Although there is no direct comparison, the AdsecVprovided better protection (80-100%) in mice than our monovalentrAd5-LcrV vaccine (˜20%) (FIGS. 3B and 4B), indicating that the secretedform of LcrV might be more immunogenic in mice. However, differentspecies of mice (Swiss-Webster versus BALB/c) and challenge doses wereused in these studies (46). In our initial study, a rAd5 expressing theIgk secretion signal fused to YFV was successfully created; however, wefound that the secreted YFV (sYFV) was toxic to HEK 293 cells, whichprevented large-scale expansion of this construct (data not shown).

There are several established plague models using NHPs, such as thelangur monkey (58), African green vervets (59, 60), baboons (61, 62),and rhesus macaque (63, 64). However, the current recommendations fromFDA and the National Institute of Allergy and Infectious Disease tosupport plague therapeutic and vaccine studies is a cynomolgus macaque(Macaca fascicularies) (CM) pneumonic plague model (65). In addition,the lethal dose of Y. pestis has been established for aerosol challengeof CMs with the standard CO92 strain, and this model was utilized inprotection studies including F1-V-based subunit vaccines for the pastseveral years as well as in most recent studies (65-72). Importantly,CMs exhibit a clinical course of the disease similar to that describedin humans (73).

Indeed, we observed the unimmunized NHPs after WT CO92 aerosol challengehad cough, respiratory changes, lethargy, and hunched posture, as wellas typical pneumonic lesions in the lungs (FIG. 14). However, no feverwas observed during the course of infection. This is in contrast to themost recent report that the onset of fever was predominant across allCMs infected with Y. pestis (72). This highlights the importance ofusing telemetry to observe physiological parameters in a real-timemanner. Our study did not employ telemetry, while the other reportmeasured body temperature in real time and the temperature of 1.5° C.above the baseline was considered fever (72). One notable finding of ourstudy was that a significant increase in the antibody titer was noted inimmunized NHPs, especially to F1, after rYFV boost as well as after WTCO92 challenge (FIG. 12 and FIG. 13). These data indicated memory B cellevoked recall responses. Similarly, a predominant hyperplasia oflymphoid follicles was observed in the immunized NHPs in mediastinallymph nodes and spleen for as long as 82 days after the WT CO92challenge (FIG. 14), suggesting a sustained immune response wasdeveloped in these NHPs, which could be pivotal in long-term protectionof animals against plague. Our studies also indicated that by using theprime-boost strategy in CMs, higher antibody responses were generatedcompared to animals that were immunized with only rAd5-YFV (FIG. 12). Anaverage antibody titers of ˜1.7×10⁶ for LcrV, ˜4.3×10⁴ for F1 and˜1.2×10⁵ for YscF, were mounted when animals were immunized followingthe prime-boost strategy. These antibodies titers were sufficient forproviding complete protection to CMs against high aerosol challengedoses of Y. pestis CO92, although the role of cell-mediated immunity inprotection should also be considered.

One of the major concerns of adenoviral vectors for vaccine developmentis the pre-existing immunity to Ad5 (in ˜95% of the human population)that could lessen the efficacy of the vaccine. Currently, most of theefforts to overcome the concerns regarding neutralizing antibodies havebeen focused on identifying alternative serotypes of adenovirus (74,75). While some groups have reported favorable results with thisapproach, it offers only a short-term solution, as new adenoviral vectoradaptation will result in the generation of neutralizing antibodiesthrough widespread use. On the other hand, a number of studies indicatedthat administration of Ad5-vectored vaccines via the i.n. route mightovercome pre-existing immunity against the Ad5 vector (76-79). We didobserve slightly lower Y. pestis antigen-specific antibody titers inmice with the pre-existing adenoviral immunity than those animalswithout the pre-existing adenoviral immunity when mice werei.n.-immunized with either the rAd5-LcrV or the rAd5-YFV vaccine (FIG.4C). However, the protection conferred in mice against Y. pestischallenge was similar in both groups of mice irrespective of thepre-existing adenoviral immunity (FIGS. 4A and 4B). Most importantly,NHPs with pre-existing adenoviral immunity and immunized with therAd5-YFV vaccine, plus a boost of rYFV, were fully protected from a highaerosol challenge dose of WT CO92 (FIG. 9).

In addition to YscF, other Y. pestis antigens such as the T3SScomponents YpkA, YopH, YopE, YopK, YopN, as well as a subunit of pH 6antigen and purified LPS were studied for their immunogenic efficaciesagainst plague infection, but did not generate promising results (80).The only protection was observed in mice vaccinated with YopD, a proteininvolved in the delivery of T3 SS effectors into the host cell (81).However, YopD-vaccination provided protection only against thenon-encapsulated bacilli but not against the encapsulated Y. pestis CO92strain.

As the most promising plague subunit vaccines currently underdevelopment are primarily dependent on only two antigens F1 and LcrV,the incorporation of a new antigen YscF may help in formulating a bettervaccine against all human plague causing-strains as we showed using thebacteriophage T4-based platform (82). Furthermore, the adenoviral vectorhas been demonstrated to have adjuvant activities as well as the abilityto promote cellular immunity (51, 83, 84). In this regard, our trivalentrAd5-YFV vaccine has unique advantages as a plague vaccine. Our furtherstudies will include in depth characterization of cell-mediated immuneresponses in vaccinated CMs.

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The complete disclosure of all patents, patent applications, andpublications, and electronically available material (including, forinstance, nucleotide sequence submissions in, e.g., GenBank and RefSeq,and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB,and translations from annotated coding regions in GenBank and RefSeq)cited herein are incorporated by reference in their entirety.Supplementary materials referenced in publications (such assupplementary tables, supplementary figures, supplementary materials andmethods, and/or supplementary experimental data) are likewiseincorporated by reference in their entirety. In the event that anyinconsistency exists between the disclosure of the present applicationand the disclosure(s) of any document incorporated herein by reference,the disclosure of the present application shall govern. The foregoingdetailed description and examples have been given for clarity ofunderstanding only. No unnecessary limitations are to be understoodtherefrom. The invention is not limited to the exact details shown anddescribed, for variations obvious to one skilled in the art will beincluded within the invention defined by the claims.

Unless otherwise indicated, all numbers expressing quantities ofcomponents, molecular weights, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless otherwise indicated to thecontrary, the numerical parameters set forth in the specification andclaims are approximations that may vary depending upon the desiredproperties sought to be obtained by the present invention. At the veryleast, and not as an attempt to limit the doctrine of equivalents to thescope of the claims, each numerical parameter should at least beconstrued in light of the number of reported significant digits and byapplying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. All numerical values, however, inherently contain a rangenecessarily resulting from the standard deviation found in theirrespective testing measurements.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

What is claimed is:
 1. A method comprising: administering a firstcomposition to a subject by an intranasal route, wherein the firstcomposition comprises a vector comprising a polynucleotide encoding afusion protein, wherein the fusion protein comprises a YscF proteindomain, a mature F1 protein domain, and a LcrV protein domain; andadministering a second composition to the subject by an intramuscularroute, wherein the second composition comprises the fusion protein,wherein the fusion protein is isolated, and wherein the intramuscularadministration is after the intranasal administration.
 2. The method ofclaim 1 wherein the fusion protein comprises at least one linker,wherein the linker is present between two of the domains.
 3. The methodof claim 1 wherein the fusion protein comprises a His-tag.
 4. The methodof claim 1 wherein the vector is a replication defective adenovirusvector.
 5. The method of claim 4 wherein the defective adenovirus vectoris type-5 (Ad5).
 6. The method of claim 1 wherein the fusion proteincomprises the YscF protein, the mature F1 protein, and the LcrV protein.7. The method of claim 1 wherein the intramuscular administration is atleast 7 days after the intranasal administration.
 8. The method of claim1 wherein the subject is a human.
 9. The method of claim 1 wherein theadministering confers immunity to plague caused by Yersinia pestis. 10.The method of claim 9 wherein the plague is pneumonic plague.